Gene Expression B: Transcription in Bacteria L12 Ch8

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• Topic: Gene Expression B – Transcription in bacteria (Chapter 1, sec. 1.2; Chapter 8).

• Core idea: Transcription in bacteria proceeds through a defined sequence of steps that convert a DNA template into an RNA transcript, guided by RNA polymerase and sigma factors.

• Key components to remember:

  • Bacterial transcription involves promoter recognition, transcription initiation, chain elongation, and chain termination.

  • The bacterial RNA polymerase core enzyme requires a sigma subunit (forming a holoenzyme) to recognize promoters.

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• Essential Steps of Transcription in E. coli:

  • Promoter recognition - occurs when the sigma subunit binds to the promoter region of the DNA, facilitating the initiation of transcription by allowing RNA polymerase to start synthesizing RNA.

  • Transcription initiation - this stage involves the formation of the transcription initiation complex, where RNA polymerase, along with the sigma factor, unwinds a short portion of the DNA double helix to expose the template strand.

  • Chain elongation- during this phase, RNA polymerase proceeds along the DNA template, adding ribonucleotides to the growing RNA strand in a complementary fashion, effectively synthesizing the RNA transcript in the 5' to 3' direction.

  • Chain termination - this process occurs when RNA polymerase encounters a termination signal in the DNA sequence, causing it to release the newly synthesized RNA transcript and detach from the DNA template.

• These four steps constitute the basic lifecycle of transcription in the cell, with regulation largely centered on promoter accessibility and sigma factor usage.

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• Bacterial RNA polymerase composition:

  • Core enzyme components:

  • α subunits (two copies) ~ $36.5 \text{kD}$ each

  • β subunit ~ $151 \text{kD}$

  • β' subunit ~ $155 \text{kD}$

  • ω subunit ~ $4 \text{kD}$ (often around 10 kD in many texts; the slide lists a small subunit weight around this range).

  • Holoenzyme: core enzyme plus a sigma (σ) subunit.

  • Function of sigma: confers promoter specificity; without sigma, the core enzyme has weak or nonspecific promoter binding.

  • Sigma diversity: there are multiple sigma factors; alternative sigma subunits give holoenzyme specificity for different promoters, enabling regulation under different conditions.

  • In E. coli, there are four main families of sigma factors described here, with their general roles:

  • σ⁷⁰ (70 kD) – housekeeping/promoters for standard growth

  • σ³² (32 kD) – heat shock genes

  • σ⁵⁴ (54 kD) – nitrogen metabolism

  • σ³⁸ (28 kD) – flagellar synthesis and chemotaxis

• Related RNA species:

  • Messenger RNA (mRNA)

  • Ribosomal RNA (rRNA)

  • Transfer RNA (tRNA)

• Note: The holoenzyme is essential for promoter recognition and initiation; the core enzyme carries out elongation.

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• The bacterial promoter architecture and promoter recognition:

  • Promoter contains two key elements upstream of the transcription start site (designated +1):

  • The -35 region with consensus sequence $ext{-}35: ext{TTGACA}$ RNA polymerase is going to bind to the promoter and initiate transcription by unwinding the DNA, thereby allowing the RNA polymerase to synthesize RNA complementary to the DNA template.

  • The -10 region (Pribnow box) with consensus starts upstream sequence $ext{-}10: ext{TATAAT}$

  • The transcription start site is designated as $+1$, and transcription proceeds downstream from there.

  • The DNA strands:

  • Coding strand (non-template) 5' → 3': $5' ext{TTGACA} ext{ ( -35 region ) } ext{ … } ext{ TATAAT } ext{ ( -10 region ) } 3'$

  • Template strand 3' → 5': $3' ext{AACTGT} ext{ ATATTA } 5'$

  • The promoter region is located upstream of the RNA-coding region; transcription start is at $+1$.

  • The 5' UTR and 3' UTR define untranslated regions of the mRNA flanking the coding sequence.

  • The promoter-box (Pribnow box) and the -35 region are recognized by RNA polymerase holoenzyme with its sigma factor.

  • What happens if transcription starts at the wrong place? The entire sequence will be off, and this can lead to the production of a nonfunctional protein or an incomplete transcript, potentially disrupting cellular processes.

  • UTR is the untranslated region

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• Transcription initiation overview: gotta have a sigma attached to the promoter region of the DNA to allow RNA polymerase to bind efficiently and commence transcription. This process ensures that the correct genes are expressed at the right times, and any errors in this initiation can cause significant issues in gene regulation.

  • The promoter region can exist in two states:

  • Closed promoter: RNA polymerase holoenzyme is bound to the promoter but DNA remains double-stranded (no open complex).

  • Open promoter: DNA around the start site is melted to form the transcription bubble, enabling RNA synthesis.

  • Start site and orientation:

  • Start site at $+1.$ There is a downstream progression from $+1$ during initiation.

  • Promoter clearance occurs after synthesis of a short RNA (often ~10–12 nucleotides), after which the enzyme transitions to the elongation phase.

  • Visual cues from the slide include labels for closed promoter, open promoter, and the start site (

  • closed promoter: recognition at -35 and -10

  • open promoter: nascent RNA begins at $+1$

  • The initiation sequence proceeds toward elongation and the transcription termination sequences define where the transcript ends.

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• E. coli RNA Polymerase Sigma Subunits (Table overview):

  • Subunits (with approximate molecular weights and function):

  • σ⁸²⁸ (σ⁽⁸²⁾) — weight ~ $28 \text{kD}$; associated with flagellar synthesis and chemotaxis (σ⁽⁶⁰⁾ family includes multiple nongenomic roles; here the slide lists 28 kD).

  • σ³² (σ⁽³²⁾) — weight ~ $32 \text{kD}$; associated with heat shock genes. When there is environmental stress is when they need to be turned on, but not always only when it is needed.

  • σ⁵⁴ (σ⁽⁵⁴⁾) — weight ~ $54 \text{kD}$; associated with nitrogen metabolism.

  • σ⁷⁰ (σ⁽⁷⁰⁾) — weight ~ $70 \text{kD}$; housekeeping genes. It is the most common in bacteria . They need all the time.

  • Consensus promoter elements per sigma factor: each sigma factor recognizes distinct promoter sequences; the canonical example is for σ⁷⁰ with -35 and -10 elements:

  • -35 consensus: $ext{-}35: ext{TTGACA}$

  • -10 consensus: $ext{-}10: ext{TATAAT}$

  • Note: The table lists the four sigma factors with their functions, illustrating how alternative sigma factors reprogram promoter recognition to adapt transcription to cellular needs.

  • need to know they function as a regulatory molecule

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• Transcription Elongation overview:

  • After initiation, the core enzyme (with the sigma factor dissociating after promoter clearance or remaining transiently) synthesizes RNA until a termination signal is encountered.

  • As RNA synthesis proceeds, the DNA duplex must unwind to provide a template strand; the transcription bubble forms and expands as nucleotides are added.

  • The coding (non-template) strand runs 5' → 3' and the template strand runs 3' → 5' within the bubble.

  • The nascent RNA extends in the 5' → 3' direction, antiparallel to the template DNA strand.

  • The promoter region is behind the open complex; beyond the start site, RNA polymerase proceeds in the 5' to 3' direction along the template strand until termination.

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• Transcription termination overview:

  • Termination occurs when the RNA polymerase encounters a termination sequence, at which point the core enzyme and nascent RNA transcript dissociate from the DNA. it only terminates under specific conditions that include the formation of a stem-loop structure in the RNA or the presence of a protein factor that guides the termination process.

  • Two major termination mechanisms in bacteria:

  • Intrinsic (rho-independent) termination

  • Rho-dependent termination

  • Both mechanisms ultimately halt transcription and release the RNA transcript.

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• Intrinsic (rho-independent) termination:

  • Features of intrinsic termination sequences:

  • Inverted repeats that can form a stem-loop (hairpin) in the RNA transcript.

  • A spacer sequence between the inverted repeats.

  • A downstream polyadenine (poly-A) sequence in the DNA template, which translates to a poly-uridine (poly-U) stretch in the RNA transcript.

  • Mechanism:

  • During transcription, the inverted repeats base-pair to form a stem-loop (hairpin) in the nascent RNA, which causes destabilization of the RNA-DNA hybrid.

  • The subsequent poly-U tract (transcribed from the template poly-A in DNA) forms weak A-U base pairs with the RNA, promoting dissociation of the RNA transcript from the DNA template.

  • Example from the slide (illustrative sequences):

  • Template DNA with inverted repeats and spacer: 5' TTATCGCCCG ACTAAATA CGGGCGATTTTTT 3'

  • Complementary template: 3' AATAGCGGGCTGATTTAT GCCCGCTAAAAAA 5'

  • Resulting RNA (mRNA): 5' UUAUCGCCCG A C UAA AU A GGGCG AUUUUU 3' (hairpin-forming region followed by a poly-U tail)

  • Base-pairing details in the hairpin region:

  • Stem: GC base pairs (strong) and CG base pairs (strong)

  • Loop: a short loop (unpaired nucleotides)

  • Termination: The transcript forms a hairpin (stem-loop) which is followed by a poly-U region that weakly pairs with the DNA template, facilitating dissociation. bacteria is ready to be translated

  • Key note: The hairpin structure itself, followed by a U-rich tail in the RNA, destabilizes the transcription complex and leads to termination.

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• Rho-dependent termination:

  • Rho utilization site (rut) on the nascent RNA marks where Rho helicase binds.

  • Rho is a hexameric RNA helicase that uses ATP hydrolysis to translocate along the RNA in the 5' to 3' direction toward the RNA polymerase.

  • Termination occurs when Rho catches up to the RNA polymerase and disrupts the transcription complex, releasing the RNA transcript.

  • The process does not require a hairpin structure in the RNA (unlike intrinsic termination); rather, it relies on Rho activity and the rut site.

  • Terminator sequences downstream ensure proper termination of transcription in coordination with Rho action.

  • Visual elements on the slide show:

  • Rut sequence on the RNA

  • Rho protein approaching the transcription complex

  • Termination sequence on the DNA and the release of mRNA

• Connections and broader points to remember:

  • Core enzyme vs holoenzyme distinction is central to promoter recognition and initiation control.

  • Sigma factors provide promoter specificity and allow rapid reprogramming of transcription in response to environmental cues (heat shock, nitrogen limitation, flagellar synthesis, etc.).

  • Promoter architecture (-35 and -10 elements) is critical for recognition by the holoenzyme and for initiation efficiency.

  • Two main termination strategies ensure proper gene expression termination: intrinsic termination using hairpin structures and poly-U tails, and Rho-dependent termination using the rut site and Rho helicase.

  • The overall process links promoter structure to initiation, elongation dynamics, and termination mechanisms, all of which are essential for understanding gene expression regulation in bacteria.